Embodiments disclosed herein relate to nucleic acids, and more particularly to methods for multiplex detection of target nucleic acid sequences.
Multiplex Ligation-dependent Probe Amplification (MLPA) is a multiplex PCR technique that permits the evaluation of, inter alia, the copy number of several target nucleic acids in a single experiment. In MLPA, each target nucleic acid queried is detected by amplification of a ligated probe. The ligated probe is generated by hybridization and ligation of a probe set comprising a pair of half-probes which are designed to reside adjacent to each other along the target nucleic acid sequence of interest. Only when the half-probes hybridize to the target nucleic acid will ligation and subsequent amplification occur.
FIG. 1 shows a flow diagram for a typical MLPA assay. The assay begins by denaturation of the DNA sample and hybridization of the probe sets to their target nucleic acid sequences. After hybridization, the adjacent half-probes are ligated and the resultant ligated probe sets are subjected to PCR amplification. The amplified ligated probe sets are then analyzed by capillary electrophoresis (CE). The peak height of the CE readout serves as the readout for the genomic target copy number.
As indicated in FIG. 1, each probe set is composed of a 5′ and 3′ half-probe having a target specific sequence and a universal primer sequence allowing the simultaneous multiplex PCR amplification of all ligated probe sets. Additionally, one (as shown in FIG. 1) or both half-probes further include a stuffer sequence which facilitates differentiation in detection of the amplified probes by CE. The use of these stuffer sequences to enable CE detection based on nucleic acid length has imposed limits on the number of target nucleic acids that may be queried in a single run. Currently, that limit is about 40 target nucleic acids. Because of the low cost, potential for high throughput, and ability to detect small rearrangements associated with MLPA, it would be beneficial to expand the number of target nucleic acid sequences that can be queried in a single run. The present disclosure provides methods for such an expansion in query number and provides related advantages as well.